Positive or plus-strand RNA viruses share many similarities in genomic organization and structure, most notably a single-stranded coding RNA of positive polarity. Representative (+) strand RNA viruses include the picornaviruses, flaviviruses, togaviruses, coronaviruses, and caliciviruses. One clinically significant representative of the flavivirus family is the hepatitis C virus (HCV), the causative agent for hepatitis C. Hepatitis C is an often chronic inflammatory disease of the liver which typically results in fibrosis and liver cancer (Choo, et al., Science (1989) 244:359). Infection by HCV typically results from contact with contaminated blood or blood products.
During HCV replication, a replicative (minus) RNA strand is produced which serves as a template for generation of several coding (+) RNA strands. The HCV genome, which contains approximately 9600 nucleotides, is translated into a polyprotein consisting of approximately 3000 amino acids (Leinbach, et al., Virology (1994) 204:163-169; Kato, et al., FEBS Letters (1991) 280:325-328). This polyprotein subsequently undergoes post-translational cleavage, producing several proteins. Due to high genetic variability and mutation rates, the HCV comprises several distinct HCV genotypes that share approximately 70% sequence identity (Simmonds, et al., J. Gen. Virol., (1994) 75:1053-1061). Despite this hypervariability, there are three regions of the HCV genome that are highly conserved, including the 5′- and 3′-non-coding regions, known as the 5′-untranslated region (5′-UTR) and 3′-untranslated region (3′-UTR), respectively. These regions are thought to be vital for HCV RNA replication as well as translation of the HCV polyprotein. In general, treatment of HCV is complicated by its high mutation rate, as well as the mode of transmission and possibility of simultaneous infection with multiple HCV genotypes.
Hepatitis C has several clinical phases. The first phase (i.e., acute phase) begins with infection by HCV. During this early phase, it is possible to detect HCV-RNA in the serum of patients using polymerase chain reaction (PCR). However, because only about 25% of patients exhibit jaundice during this phase, most cases (75%) go undetected in the early stages. The inflammatory process, characterized by an increase in serum liver enzyme concentrations, begins approximately four weeks post infection. Although acute HCV infection is not malignant, the majority of patients (approximately 80%) develop chronic liver disease, characterized by a permanent elevation in the serum alanine aminotransferase level. Cirrhosis of the liver develops in more than 20% of patients with chronic HCV disease, which frequently leads to malignant hepatoma. Life expectancy following diagnosis of the malignant hepatoma is generally 12 months.
Current therapies to treat HCV infections have met with limited success, with only a minority of patients experiencing long-term improvement. The most prevalent treatment today involves specific cytokines known as interferons, particularly interferon-α (IFN-alpha) which reduces serum alanine aminotransferase levels in approximately 50% of patients. Unfortunately, serum levels of alanine aminotransferase usually return to elevated levels following termination of treatment, producing a number of adverse side effects (Dusheiko, et al., J. Viral Hepatitis (1994) 1:3). Despite these problems, IFN-alpha is commonly used to reduce the risk of cirrhosis of the liver and malignant hepatoma. There is no currently available vaccine for HCV.
Although IFN-alpha remains the conventional approach, virologists have evaluated a number of potential alternative therapies, including the use of specific ribozymes to inhibit translation of viral protein. Welch et al. disclose a two vector-expressed hairpin ribozyme directed against HCV (Welch, et al., Gene Therapy (1996), 3(11):994). Lieber et al. report the removal of HCV-RNA in infected human hepatocytes through adenovirus-mediated expression of specific hammerhead ribozymes (Lieber, et al., Virology (1996), 70 (12):8782). WO 99/55847 reports the degradation of 5′- and 3′-UTL regions of HCV-RNA, as well as the 5′-coding region for the nucleoprotein, using ribozymes. U.S. Pat. No. 5,610,054 discloses enzymatic nucleic acid molecules that can inhibit replication of HCV. Despite these efforts, the therapeutic value of ribozymes for treating HCV infections remains questionable, particularly in view of their low enzymatic activity.
More recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). Briefly, the RNAse III Dicer processes dsRNA into small interfering RNAs (siRNA) of approximately 22 nucleotides, which serve as guide sequences to induce target-specific mRNA cleavage by an RNA-induced silencing complex RISC (Hammond, S. M., et al., Nature (2000), 404:293-296). When administered to a cell or organism, exogenous dsRNA has been shown to direct the sequence-specific degradation of endogenous messenger RNA (mRNA) through RNAi. WO 99/32619 (Fires et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of a target gene in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.); Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200); and mammals (WO 00/44895, Limmer).
Despite significant advances in the field, there remains a need for an agent that can inhibit the replication of a virus in a host cell using the cell's own RNAi machinery. More specifically, an agent that has high biological activity and can provide long-term, effective inhibition of viral replication at a low dose would be highly desirable. Compositions comprising such agents would be useful for treating a variety of viral infections, including HCV.